CMP-A -acetylneuraminic acid

This enzyme [EC 2.7.7.43], also referred to as CMP-A-acetylneuraminic acid synthetase and CMP-sialate synthase, catalyzes the conversion of CTP and A-acylneu-raminate to yield pyrophosphate and CMP-A-acylneu-raminate. The protein will act on both the A-acetyl and A-glycolyl derivatives. 

Figure 2.19 Sialylation ofN-acetyl lactose by cytidyl monophosphate-N-acetylneuraminic acid using Of 2,3-neuraminic acid transferase as catalyst (upper box). Regeneration of the sugar nucleotide is shown in the lower box. CMP is converted into CTP in two steps using two different kinases. In the final step CMP-A -acetylneuraminic acid is synthesised from CTP and A -acetylneuraminic acid (sialic acid) using the appropriate synthetase. The formed pyrophosphate is converted into inorganic phosphate. Altogether five different enzymes are involved in the process.

Adenosine 5 -phosphosulfate 3 -Phosphoadenosine 5 -phosphosulfate CMP-A-acetylneuraminic acid... 

The carbohydrate portion of glycoproteins is formed from UDP-iV-acetylglucosamine and CMP-A-acetylneuraminic acid, and is attached to the protein by A-glycosidic bonds to asparagine amide groups. 

Kinetic parameters for the CMP-A -acetylneuraminic acid glycoprotein sialyltransferase in human malignant melanoma have been reported. An overall scheme for the metabolism of acylated neuraminic acids in animal tissues, based on a study of 0-acylation of the sialic acid residues in bovine and equine submandibular glands, has been presented. ... 

The 5-N-acetyl group of CMP-A -acetylneuraminic acid can be modified by a hydroxylase to form CMP-7V-glycolyl-neuraminic acid. The enzyme occurs in many non-human tissues and has been cloned from several species [113]. JV-Glycolyl-neuraminic acid can then be transferred by sialyltransferases to glycoproteins and mucins, resulting in a sialic acid modification which is common in manunalians but in humans is antigenic, and only found in exceptional conditions such as in cancer (5). 

The biosynthetic pathway for sialic acid formation in liver was well established nearly thirty years ago, by the elegant work from the groups of Roseman [9-11] and Warren [12] (Figure 1). The enzymatic conversion of UDP-A-acetyl glucosamine to A-acetylmannosamine is the critical step in the overall synthesis of sialic acid because the activity of this epimerase is controlled downstream in the pathway by feedback inhibition by CMP-A -acetylneuraminic acid [13]. Thus, production of ManNAc as a precursor for sialic acid biosynthesis is a closely regulated cellular process. 

In order to try to overcome some of the problems associated with chemical synthesis of oligosaccharides containing N-acetylneuraminic acid, Sabesan and Paulson [277] have used a combination of chemical and enzymatic methods using purified sialyl-transferases in the presence of CMP-iV-acetylneuraminic acid and synthetic acceptor molecules to give sialyl derivatives of oligosaccharides which were characterised by NMR. Thus, methyl P-D-galactopyranoside, methyl P-D-lactoside and iV-acetyl-... 

HeLa cells were cultured for 24 hr with (open bars) and without (closed bars) 5 mM sodium butyrate, harvested, and assayed for indicated enzyme activity. A GM3-sialidase activity assayed with GM3 specifically labeled with [,iC]- N-acetylneuraminic acid as substrate ("14JJ. B Sialyltrasferase activity assayed with CMP-[, acetylneuraminic acid and lactosylceramide as substrates, and synthesis of labeled GM3 determined (17). Data... 

UDP-D-xylose, GDP-D-mannose, GDP-L-Fucose, or CMP-D-A-acetylneuraminic acid. Mammalian GTs also use dolichol-diphosphate-GlcNAc2Man9GlC3, dolichol-phosphate-mannose and dolichol-phosphate-glucose (Fig. 2). 

In contrast to other monosaccharides, activated sialic acid donors are biosynthesized from A -acetylmannosamine (ManNAc) or directly from sialic acids (Sia), including A-acetylneuraminic acid (NeuAc), via a more complex pathway (21). ManNAc is phosphorylated at the at the 6-hydroxyl group and condensed with phosphoenolpyruvate to give A -acetylneuraminic acid-9-phosphate (NeuAc-9-P). Phosphate ester hydrolysis is followed by direct condensation with CTP to give CMP-NeuAc (Figure 3). Sialic acids can intercept this pathway directly via enzymatic reaction with CTP. 

Fig. 15. Biosynthesis of CMP-A -glycolylneuraminic acid with the aid of CMP-At-acetylneuraminic acid monooxygenase in the cytosol, translocation of CMP-Neu5Gc into the Golgi vesicle and transfer of the NeuSGc moiety onto nascent glycoconjugates [188,654,684]. From ref [690] by permission of Oxford University Press, Oxford.

CMP-N-acetylneuraminic acid (CMP-NeuAc). CMP-N-acetylneuraminic acid has been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc, under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43) [131l An improvement in this procedure, involving in situ production of CTP from CMP under adenylate kinase and pyruvate kinase catalysis, is suitable for multigram-scale synthesis11321. Adenylate kinase catalyzes the equilibration of CTP and CMP to CDP, which is subsequently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of CMP-NeuAc based on this procedure involves the in situ synthesis of NeuAc from N-acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[10S1. Chemical syntheses of CMP-NeuAc have also been reported11421. 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which A-acetylneuraminic acid (NeuAc) is generated in situ from JV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

To HEPES buffer (100 mL, 200 mAf, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), A-acetylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H " form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

A similar example is the biosynthesis of UDP-2-acetamido-2-deoxy-D-glucose and CMP-N-acetylneuraminic acid in rat liver.Here again, both sugars are needed for syntliesis of complex saccharides, and each sugar nucleotide inhibits the first enzyme of the pathway unique to its synthesis, as shown by the dotted arrows in the following Scheme. 

Escherichia coli K-235 produces an extracellular homopolymer of AT-acetylneuraminic acid. This substance, named colominic acid, has been synthesized in vitro from CMP-AT-acetylneuraminic acid in the presence of a purified particulate fraction from this strain of E. coli. The activity of the enzyme is stimulated by ammonium sulfate (1.2-1.5M), and by the addition of exogenous colominic acid (especially of aged, slightly degraded preparations). 

CMP-acylneuraminate synthase Reaction with agarose cyclic imido-carbonate Active immobilized enzyme synthesis of CMP-A-acetylneuraminic and CMP-A-glycoloylneuraminic acids 122... 

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